Mechanisms of post-translational regulation of podoplaninProteolysis and phosphorylation

Resumen

Podoplanin is a small mucin-like transmembrane glycoprotein that plays an important role in the development of the heart, the lungs, and the lymphatic system. Its expression has been shown to correlate with cancer progression, increased lymphatic invasion and nodal metastasis, and poor clinical outcome. Despite the growing knowledge on the function of podoplanin, both physiologically and pathologically, little is known regarding its regulation. Here, we report that podoplanin expression is regulated at the post-translational level in several normal and tumour cell lines by a novel calpain-dependent mechanism. Calpain digestion is, however, only the initial proteolytic step that primes podoplanin for complete degradation by a still unidentified degradation pathway. In addition, we provide evidence for the occurrence of two new post-translational modifications of podoplanin. Podoplanin undergoes a constitutive sequential proteolytic processing that involves the cleavage of its extracellular domain by a metalloprotease, and the subsequent cleavage of the membrane-bound C-terminal fragment (PCTF) by gamma-secretase which releases podoplanin's intracellular domain (PICD) fragment. We also demonstrate for the first time that podoplanin is being phosphorylated. Contrary to what had been predicted, podoplanin does not seem to be a substrate for PKA or PKC, but is constitutively phosphorylated by Cdk5 at S161. Importantly, the main phosphorylation band corresponds to PICD, and phosphorylation appears to act as a mechanism preventing the nuclear localization of this fragment. The exact function of podoplanin's proteolytic processing and phosphorylation has not been determined yet, but S161 seems a key amino acid in the regulation of podoplanin function. Indeed, contrary to wild-type podoplanin, the phosphomimetic mutant and podoplanin-deltaYS isoform, which lacks two residues in its cytoplasmic domain including S161, cannot induce ERM (ezrin, radixin, moesin) protein activation and an epithelial to mesenchymal transition (EMT) in Madin-Darby Canine kidney (MDCK) cells.